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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
1Department of Physiology and Institute for Biomedical Research, The University of Sydney, Sydney, New South Wales 2006, Australia; and 2Department of Physiology, National Defense Medical College, Tokorozawa, Saitama 359-6518, Japan
Submitted 20 April 2004 ; accepted in final form 20 July 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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20 mmHg in the MAP but had no effect, when compared with the effect of vehicle injection alone, on HR or RSNA. By 50 min after the injections of Kyn or vehicle in the RVLM, the MAP had stabilized at a level close to its original baseline level, but the HR and RSNA stabilized at levels above baseline. The results indicate that removal of tonic EAA drive to RVLM neurons has little effect on the tonic activity of RVLM presympathetic neurons, even when inputs from the CVLM are blocked. Thus the tonic activity of RVLM presympathetic neurons under these conditions is dependent on excitatory synaptic inputs mediated by non-EAA receptors and/or the autoactivity of these neurons.
glutamate; 
-aminobutyric acid; renal sympathetic nerve activity; caudal ventrolateral medulla; excitatory amino acid; rostral ventrolateral medulla
The mechanisms that underlie the resting tonic activity of RVLM presympathetic neurons, however, are still unclear. As we have reviewed recently (9), there is evidence that these neurons receive tonically active synaptic inputs under resting conditions and, in addition, may be capable of generating intrinsic activity, at least under abnormal conditions when all synaptic inputs are blocked. With regard to tonic synaptic inputs, it is well accepted that these neurons receive a tonic GABAergic input, because blockade of GABAA receptors in the RVLM results in an increase in both arterial pressure and sympathetic vasomotor activity (8, 20). This tonic GABAergic inhibition is believed to originate to a large extent from neurons within the caudal ventrolateral medulla (CVLM) and is driven by both baroreceptor-dependent and baroreceptor-independent inputs (5, 6, 8). In contrast to GABA receptors, however, blockade of ionotropic EAA receptors by microinjection of kynurenic acid (Kyn) in the RVLM of the rat has little effect on arterial pressure or sympathetic vasomotor activity (10, 13, 24), which has led to the suggestion that these neurons do not receive a significant tonic excitatory input mediated by EAA receptors, at least in the rat.
On the other hand, there is evidence that RVLM neurons receive tonic excitatory synaptic inputs, as indicated by intracellular recordings from identified spinally projecting barosensitive neurons within the region (17). Furthermore, Ito and Sved (13) have reported that, after inhibition of the CVLM by local injection of the GABAA receptor agonist muscimol, subsequent blockade of ionotropic EAA receptors (by microinjection of Kyn) in the RVLM does cause a profound fall in arterial pressure in anesthetized rats. To explain their observation that Kyn microinjections in the RVLM caused a profound fall in arterial pressure under these conditions, whereas they have little effect under normal baseline conditions, Ito and Sved (13) proposed that a Kyn-sensitive input to the RVLM is tonically active under normal baseline conditions and provides both an excitatory input to RVLM presympathetic neurons and an excitatory input to neurons in the CVLM, which then provide a counterbalancing inhibition of RVLM presympathetic neurons. Thus, according to this model, under normal conditions blockade of EAA receptors in the RVLM will result in removal of both tonic excitatory and inhibitory influences on RVLM presympathetic neurons, resulting in little or no net change in sympathetic vasomotor activity and arterial pressure. The model proposed by Ito and Sved (13) also included a separate tonic excitatory input from the CVLM to the RVLM mediated by non-EAA receptors, to account for the fact that RVLM presympathetic neurons are tonically active after blockade of EAA receptors within the RVLM.
One potential difficulty in the interpretation of the results presented by Ito and Sved (13), however, is that bilateral inactivation of neurons in the CVLM causes strong sympathoexcitation (5, 6), which would be expected to cause intense vasoconstriction. This, in turn, will decrease venous return and cardiac output, so that under those circumstances the changes in arterial pressure that occur after muscimol injections in the CVLM may not necessarily reflect changes in sympathetic vasomotor activity. The primary aim of this study, therefore, was to determine the effect on renal sympathetic nerve activity (RSNA) of bilateral blockade of glutamate receptors in the RVLM after bilateral inhibition of the CVLM.
As mentioned above, tonic inhibitory inputs from the CVLM to the RVLM are believed to be mediated via GABA receptors (2, 5, 7, 10). Thus, according to the model proposed by Ito and Sved (13), after blockade of GABA receptors in the RVLM the tonic activity of RVLM presympathetic neurons would be maintained by both EAA and non-EAA excitatory inputs. Therefore, a second aim of the study was to determine the contribution of EAA receptors in the RVLM to the maintenance of sympathetic vasomotor activity under conditions where GABAergic inhibition of RVLM neurons has been blocked.
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METHODS |
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The rat was fixed in a stereotaxic apparatus, and the incisor bar was set at 19 mm below the interaural line. The dorsal surface of the medulla was exposed, and microinjections were made in sites within the medulla using a micropipette held in a micromanipulator at an angle of 20° (tip rostral). The compounds injected were sodium glutamate (10–20 nl of 50 mM solution; Sigma), muscimol (100 nl of 2 mM solution; Sigma), Kyn (100 nl of 27 mM solution in all experiments, except for 3 experiments in which 200 nl of 27 mM solution was injected; Sigma), and bicuculline methochloride (100 nl of 2 mM solution; Tocris). The vehicle solution for injections of sodium glutamate and muscimol was artificial cerebrospinal fluid (aCSF) adjusted to pH 7.4. To prepare the Kyn solution, Kyn was first dissolved in the minimum amount of 1 N NaOH solution, and then aCSF was added to the dissolved solution, after which the pH was adjusted to 7.4 by addition of 0.1 N HCl. When injections of vehicle were made instead of Kyn in the RVLM, the vehicle solution used was aCSF with the same amounts of NaOH and HCl added as was used to make up the Kyn solution. Injections were made by pressure, and the volume injected was measured by the displacement of the meniscus in the pipette with respect to a horizontal grid viewed through an operating microscope.
The pressor region within the RVLM on each side was identified as the site at which microinjection of sodium glutamate evoked a pressor response of at least 20 mmHg and an increase in RSNA of at least 40% with respect to the preinjection level. The coordinates of the pressor site in the RVLM were 0.8–1.0 mm rostral to the calamus scriptorius (at the point of entry of the pipette at the dorsal surface), 2.0 mm lateral to the midline, and 3.0–3.2 mm below the dorsal surface. Similarly, the depressor region in the CVLM on each side was identified as the site at which microinjection of sodium glutamate evoked a depressor response of at least 25 mmHg and a decrease in RSNA of at least 50% with respect to the preinjection level. The coordinates of the depressor site in the CVLM were 0.5 mm caudal to the calamus scriptorius (at the point of entry of the pipette at the dorsal surface), 1.8–2.0 mm lateral to the midline, and 2.8 mm below the dorsal surface. Usually no more than two injections were required to identify pressor or depressor sites in the RVLM and CVLM, respectively.
After the identification of the RVLM and CVLM injection sites as described above, there was a waiting period of >20 min to allow for the MAP, HR, and RSNA to stabilize. In the first series of experiments, bilateral microinjections of muscimol (2–4 min apart) were made in the CVLM depressor region. Once the RSNA had reached a peak level after the muscimol injections, bilateral injections of Kyn (3–4 min apart) were made in the RVLM pressor region. The MAP, HR, and RSNA then continued to be recorded until these variables had stabilized at new resting levels (50 min after the Kyn injections). In control experiments, the procedure was the same except that the vehicle solution instead of the Kyn solution was injected in the RVLM.
In two experiments, before the start of the experimental procedure described above, the somato-sympathoexcitatory reflex was tested before and after bilateral microinjections of Kyn (100 nl of 27 mM solution) in the RVLM pressor region, as a test of the effectiveness of the blockade of EAA receptors in the RVLM, which are known to mediate the somato-sympathoexcitatory reflex (14). In these experiments, the sciatic nerve was exposed, placed on bipolar stimulating electrodes, and immersed in a pool of mineral oil. The somato-sympathoexcitatory reflex was tested by recording the reflex increase in RSNA evoked by electrical stimulation of the sciatic nerve, according to the procedure described previously (24). The magnitude of the stimulus (square wave pulse, 20–25 V, 1-ms duration) was chosen such that it elicited a large but submaximal response.
In three experiments, bilateral injections of muscimol were made in the more rostral part of the CVLM [i.e., 0.1 mm caudal to the calamus scriptorius (at the point of entry of the pipette at the dorsal surface), 1.8–2.0 mm lateral to the midline, and 2.8 mm below the dorsal surface]. In all three of these experiments, a microinjection of glutamate was first made in these sites, and in all three cases this injection evoked a significant depressor and sympathoinhibitory response, as described above.
In the second series of experiments, the procedure was the same as in the first series except that bilateral microinjections of bicuculline were first made in the RVLM, instead of bilateral microinjections of muscimol in the CVLM. In addition, control experiments were also performed for this series, in which the procedure was the same except that the vehicle solution instead of the Kyn solution was injected in the RVLM after the bilateral microinjections of muscimol in the CVLM.
At the end of each experiment, the centers of the injection sites of muscimol, Kyn, bicuculline, and vehicle solution in the RVLM or CVLM were marked by microinjection in the same site of aCSF containing 1% Fast Blue (100 nl). The rat was then killed with an overdose of pentobarbital sodium. The brain was then removed and placed in 50 ml of 4% paraformaldehyde solution for 1–3 days, after which 50-µm-thick sections were cut on a freezing microtome and mounted on glass slides. Injection sites were determined using a fluorescence microscope and were mapped on to standard sections according to the atlas of Paxinos and Watson (19). The rostrocaudal level of the injection sites in the RVLM or CVLM section was determined by examination of the shape and size of the ventrally located landmarks close to the injection sites (e.g., caudal pole of the facial nucleus and nucleus ambiguus for the RVLM, and lateral reticular nucleus, inferior olive, and nucleus ambiguus for the CVLM). For injection sites in the CVLM, the area postrema was also used as a dorsally located landmark.
The baseline values for MAP, HR, and RSNA were measured as the average values for each of these variables over the 30-s period immediately preceding bilateral injections of muscimol in the CVLM, or of bicuculline in the RVLM. The magnitudes of the changes in MAP, HR, or RSNA at different times after injections of Kyn in the RVLM were compared with those that followed injections of vehicle solution in the RVLM by a repeated-measures ANOVA. Comparisons of the changes in MAP and RSNA evoked by microinjections of Kyn or vehicle in the RVLM were made using an unpaired t-test. A P value of <0.05 was regarded as statistically significant. All values are presented as means ± SE.
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RESULTS |
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50 min that was very similar to the preinjection baseline level (Figs. 1 and 2). In contrast, after the vehicle injections, the MAP initially decreased more slowly, but eventually reached a final stable level that was also very similar to the preinjection baseline level (Fig. 2). The decrease in MAP during the first 5 min postinjection was significantly greater in the experiments in which Kyn was injected (63 ± 7 mmHg from a level of 175 ± 8 mmHg just before Kyn injection) compared with the experiments in which vehicle was injected (21 ± 10 mmHg from a level of 158 ± 12 mmHg just before vehicle injection, P < 0.05).
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The increase in HR that occurred after muscimol injections in the CVLM was sustained throughout the period of recording and was not altered by subsequent bilateral injections of either Kyn or vehicle solution (Figs. 1 and 2).
In three additional experiments, the procedure was the same except that the injection volume of the Kyn solution was 200 instead of 100 nl. In these experiments, the pattern of changes in MAP and RSNA after bilateral injections of 5.4 nmol Kyn in 200 nl solution was similar to that observed when 2.7 nmol Kyn in 100 nl solution was injected. After bilateral injections of muscimol in the CVLM, the MAP increased to 160 ± 11 mmHg from a baseline level of 80 ± 12 mmHg. After subsequent bilateral injections of Kyn in the RVLM, the MAP first decreased rapidly to 84 ± 9 mmHg (by 5 min after the Kyn injections), but then increased gradually back to a level of 101 ± 9 mmHg by 30 min after the Kyn injections. The RSNA also increased after bilateral injections of muscimol in the CVLM (to 258 ± 53% of the baseline level). After subsequent bilateral injections of Kyn in the RVLM, the RSNA decreased to a small extent (to 212 ± 47% of the baseline level by 5 min after the Kyn injections) but then increased again gradually back to a level of 250 ± 59% by 30 min after the Kyn injections.
Histological analysis confirmed that the centers of the injection sites for both Kyn and vehicle injections were located within the RVLM, i.e., at the level of the caudal pole of the facial nucleus or more caudally, within 0.5 mm of the caudal pole [i.e., close to the level 12.30 mm caudal to bregma, according to the atlas of Paxinos and Watson (19), Fig. 3]. The centers of the injection sites for muscimol were located within the CVLM [close to the level corresponding to the level 13.8 mm caudal to bregma, according to the atlas of Paxinos and Watson (19), Fig. 3].
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In three experiments, bilateral microinjections of muscimol were made in the more rostral part of the CVLM, as described in METHODS. In these experiments, no subsequent injections were made in the RVLM. In all three of these experiments, muscimol microinjections resulted in initial marked increases in MAP, HR, and RSNA. The MAP reached a peak level 1–3 min after the second injection, whereas the HR and RSNA reached peak levels 3–8 min after the second injection (Fig. 4). Subsequently, however, the RSNA declined (Fig. 4), at a more rapid rate than in the experiments in which muscimol was injected in the more caudal part of CVLM, and eventually stabilized after 50 min at a level that was well below the preinjection baseline level (55 ± 3% baseline level). Similarly, the MAP also declined to a final resting level (57 ± 9 mmHg) that was also well below the preinjection baseline level (96 ± 4 mmHg). For these three experiments, the centers of the muscimol injection sites were located at a rostrocaudal level that was judged from histological examination to be close to 13.4 mm caudal to bregma, according to the atlas of Paxinos and Watson (19; Fig. 4).
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20 mmHg in three of five experiments, but this was not associated with any significant change in RSNA (e.g., Fig. 5). Bilateral microinjections of the vehicle solution did not have any immediate effect on MAP or RSNA in any experiment. Overall, however, the time courses and magnitudes of changes in MAP, HR, and RSNA after either Kyn or vehicle injections in the RVLM were very similar (Fig. 6).
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DISCUSSION |
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An important question is the extent to which the muscimol injected in the CVLM blocked the activity of GABAergic neurons that tonically inhibit RVLM presympathetic neurons, as well as the putative interneurons in the CVLM that, according to the proposed model of Ito and Sved (13), provide a tonic excitatory input to the RVLM presympathetic neurons. A related question is the extent to which Kyn injected in the RVLM blocked EAA receptors on the RVLM presympathetic neurons. In considering these questions, it must be borne in mind that there is no clear separation between presympathetic neurons in the RVLM and the sympathoinhibitory interneurons in the CVLM. As demonstrated in detail by Weston et al. (25), there is significant overlap between these two populations of neurons. Previous studies in which c-Fos labeling was used to identify populations of medullary barosensitive neurons in the rat (4) and rabbit (16) also demonstrated an overlap in the rostrocaudal distribution of sympathoexcitatory and sympathoinhibitory neurons in the ventrolateral medulla. Thus it is not possible to inhibit all sympathoinhibitory neurons in the CVLM by means of microinjection of muscimol without affecting RVLM presympathetic neurons. This was illustrated by the finding that, when muscimol was injected in the more rostral part of the CVLM, the MAP and RSNA did gradually decline to levels that were well below the baseline levels (to 57 ± 9 mmHg and 55 ± 3% baseline, respectively), even though in these experiments Kyn was not injected in the RVLM. In this case, the hypotensive and sympathoinhibitory effect is presumably the result of diffusion of muscimol to sympathoexcitatory neurons within the RVLM. Such an effect, however, is not likely to account for the decrease in MAP observed by Ito and Sved (13) when Kyn was injected in the RVLM after injections of muscimol in the CVLM, because in their study bilateral injections of muscimol in the CVLM, without subsequent injections of Kyn into the RVLM, resulted in a sustained increase in arterial pressure.
Histological examination was used to identify the centers of the injection sites in the CVLM. Although the rostrocaudal level [with respect to the atlas of Paxinos and Watson (19)] of the dorsal part of each section could be accurately determined, using the calamus scriptorius and area postrema as landmarks, there were potential errors in the determination of the rostrocaudal level of the injection sites in the CVLM, as a consequence of variation in the angle of cut of the sections. Thus, even though the shape and size of nearby ventrally located landmarks (see METHODS) were used as a further guide, we estimate that there was a possible error of up to ±0.2 mm in the estimate of the rostrocaudal level of the injection sites in the CVLM. The actual level of the injections, therefore, would have been in the range of 13.6–14.0 mm caudal to bregma [with respect to the atlas of Paxinos and Watson (19)]. These levels correspond to the caudal part of the group of baroactivated GABAergic neurons as mapped by Weston et al. (25). It is likely, however, that the muscimol would have diffused to more caudal parts of the CVLM that contain neurons that are not baroactivated but which also project to and inhibit RVLM presympathetic neurons (6).
Although there is likely to have been some variability in the precise locations of the muscimol injection sites in the CVLM, in all cases these injections resulted in large increases in both MAP and RSNA. These increases were of very similar magnitude to the increases in these variables evoked by bilateral injections of bicuculline in the RVLM, which would be expected to block all tonic inhibitory GABAergic inputs to the RVLM arising from the CVLM and other sources. Thus the results indicate that the muscimol injections blocked at least a large part of the tonic GABAergic inhibitory input to RVLM presympathetic neurons, although it cannot be concluded that this blockade was necessarily complete in all cases.
According to the model proposed by Ito and Sved (13), RVLM presympathetic neurons also receive a tonic non-EAA excitatory input originating from the CVLM, to account for the fact that RVLM presympathetic neurons are tonically active after blockade of EAA receptors within the RVLM. Thus one possible explanation for the differences in our findings compared with those of Ito and Sved, is that in our study the muscimol injection sites in the CVLM had a different location to those in the study by Ito and Sved, and thus may not have inhibited these putative sympathoexcitatory neurons in the CVLM. It is difficult to compare the precise locations of the muscimol injection sites in the CVLM in our study with those in the study by Ito and Sved (13), because the latter study did not provide information about the histological localization of the CVLM injection sites. In both studies, however, the volume and concentration of the muscimol injections were the same (100 nl of 2 mM solution), and in both cases were made into functionally identified depressor sites. Furthermore, in both our study and that of Ito and Sved (13), the muscimol injections in the CVLM evoked increases in MAP of similar magnitude. It therefore seems unlikely that, in our study, the locations of the muscimol injection sites in CVLM were so different from the injection sites in the study by Ito and Sved (13) that they failed, in all experiments, to inhibit the putative CVLM neurons that provide a crucial non-EAA tonic excitatory input to RVLM presympathetic neurons.
The dose of Kyn injected in the RVLM (2.7 nmol) has been shown previously to block the somato-sympathoexcitatory reflex (15), which is mediated by non-NMDA ionotropic EAA receptors (14), and this was confirmed in the present study in two experiments. It has also been shown that this dose is also sufficient to block pressor responses elicited by microinjection of selective agonists of both NMDA and non-NMDA ionotropic EAA receptor subtypes in the RVLM (15). Nevertheless, it is possible that, after the bilateral injections of 100 nl Kyn solution, some tonic glutamatergic input to RVLM presympathetic neurons remained, which was sufficient to maintain sympathetic vasomotor tone. Even when a much larger volume (200 nl) of Kyn solution was injected in the RVLM after bilateral muscimol injections in the CVLM, the RSNA still remained at a high level, well above the preinjection baseline level, for at least 50 min after the Kyn injections.
In summary, although we cannot rule out the possibility that, in some experiments, the muscimol injections in the CVLM failed to block a putative non-EAA tonic excitatory input to RVLM presympathetic neurons or that the Kyn injections in the RVLM failed to block all tonic EAA excitatory inputs, the fact remains that, in all the experiments in which muscimol was injected bilaterally in the CVLM followed by bilateral Kyn injections in the RVLM, a profound decrease in MAP similar to that described by Ito and Sved (13) was never observed, except (as discussed above) when the muscimol injections were made in the more rostral part of CVLM. Furthermore, in all experiments, the RSNA remained at levels well above the preinjection baseline level. Our findings therefore do not provide any evidence to support the hypothesis that sympathetic vasomotor tone is dependent on a tonic EAA-mediated excitatory input to RVLM presympathetic neurons, under conditions where a tonic excitatory input to those neurons from the CVLM is eliminated.
There were, however, some differences in the experimental conditions between our study and that of Ito and Sved (13) that need to be considered. First, in the study by Ito and Sved the rats were initially anesthetized with halothane (before replacing the halothane with either iv chloralose or urethane), whereas in our study the rats were anesthetized initially with intraperitoneal urethane. Second, in our study, the extent of the surgery was greater in that the renal nerve was exposed to enable the recording of RSNA. Third, the baseline levels of MAP in our study (90 mmHg) were less than in the study by Ito and Sved (120 mmHg). Thus it is possible that there were differences between our study and that of Ito and Sved (13) with respect to the degree of tonic excitatory input to the RVLM presympathetic neurons, which might have resulted in differences in the level of sympathetic vasomotor tone. At the same time, it should be noted that the increase in MAP evoked by bilateral injections of muscimol in the CVLM were of similar magnitude in the two studies, suggesting that the responsiveness of the RVLM presympathetic neurons to blockade of tonic GABAergic inputs was similar in the two studies. In summary, although there were some differences in the anesthetic conditions, extent of surgery, and baseline levels of MAP between our study and that of Ito and Sved (13), it is not clear how such differences could account for the fact that, in our study, glutamate receptor blockade in the RVLM after CVLM inhibition did not result in a profound hypotension, whereas this was observed in the study by Ito and Sved (13).
Our results do indicate, however, that EAA receptors make some contribution to maintaining the high level of arterial pressure, but not the increased RSNA or HR, that occurs as a consequence of inhibition of the CVLM. Similarly, we also found that, after bilateral microinjections of bicuculline in the RVLM, thus blocking tonic GABAergic inhibition of RVLM neurons, subsequent microinjections of Kyn in the RVLM resulted in a modest decrease in MAP of 20 mmHg in three of five experiments, which in this case also was not accompanied by a significant change in RSNA. A possible reason for this difference in the effects of Kyn injections on MAP and RSNA is that the tonic activity of RVLM neurons that regulate the sympathetic outflow to nonrenal vascular beds (e.g., skeletal muscle or splanchnic beds) may be more dependent on EAA receptor-mediated inputs than is the case for RVLM neurons regulating the renal sympathetic outflow. Consistent with this possibility, Miyawaki et al. (18) found that, after bicuculline injections in the RVLM, subsequent injections of Kyn in the RVLM resulted in a decrease in MAP to a level 20% below the peak postbicuculline level, which was accompanied by decreases in lumbar and splanchnic sympathetic nerve activity to levels 10–20% below their respective postbicuculline peak levels.
In all experiments in which muscimol was injected in the CVLM and then either Kyn or vehicle injected in the RVLM, the RSNA decreased slowly from its peak level, but eventually stabilized at a level that was still increased with respect to the preinjection baseline level. In contrast, the MAP eventually stabilized at a level that was very close to its preinjection baseline level. A possible explanation for this difference is that, under conditions in which there is intense widespread vasoconstriction, as a result of inhibition of the CVLM, the venous return and thus cardiac output will decrease. Second, the initial large increase in MAP and hence cardiac afterload may also lead to a subsequent reduction in stroke volume that would contribute to the decline in MAP. Thus, at the time when the cardiovascular variables have stabilized, the sympathetic vasomotor tone and total peripheral vascular resistance would be increased, but the cardiac output decreased, such that the MAP is changed little. In any case, the results demonstrate that the changes in MAP that occur after injections of Kyn in the RVLM do not necessarily reflect changes in RSNA.
In the experiments in which bicuculline was injected bilaterally in the RVLM, the dose used (200 pmol) has been shown previously to be sufficient to abolish baroreflex sympathoinhibition (21, 24), which is mediated by GABA receptors in the RVLM (10, 20). Thus it is likely that this dose produced complete or nearly complete blockade of GABA receptors in the RVLM. Even under these circumstances, in which GABAergic inputs to RVLM neurons from all sources are blocked, subsequent bilateral injections of Kyn in the RVLM had little effect (compared with vehicle injections) on the MAP or RSNA, apart from a modest decrease in MAP of 20 mmHg that occurred in some experiments, as discussed above. Thus the results of the present study demonstrate that, even after blockade of GABA receptors in the RVLM, subsequent blockade of EAA receptors in this region has little effect on RSNA. As mentioned above, Miyawaki et al. (18) found that, under the same experimental conditions, subsequent blockade of EAA receptors in the RVLM resulted in a significant decrease in lumbar and splanchnic sympathetic nerve activity (of 10–20% of the peak levels after bicuculline injections). In both our study and that of Miyawaki et al. (18), however, after blockade of GABA receptors in the RVLM followed by blockade of EAA receptors in the RVLM, the MAP and sympathetic vasomotor activity were still increased with respect to the initial baseline levels. These results are also consistent with those of a previous study from our laboratory in which we found that blockade of EAA receptors followed by blockade of GABA receptors in the RVLM resulted in an increase in MAP and RSNA (24).
Under conditions when both EAA and GABA receptors in the RVLM are blocked, the tonic activity of presympathetic neurons in this region must be dependent either on non-EAA excitatory synaptic inputs and/or the autoactivity of the RVLM neurons, as originally suggested by Sun et al. (22). If non-EAA excitatory synaptic inputs to RVLM neurons are essential for maintaining sympathetic vasomotor tone, the results of the present study indicate that they arise, at least in part, from regions other than the CVLM.
At the same time, our observations do not preclude the possibility that RVLM neurons receive tonic synaptic excitatory inputs mediated by EAA receptors under normal conditions. As pointed out by Lipski et al. (17), it is possible that under normal conditions the activity of RVLM presympathetic neurons is determined by the balance of excitatory and inhibitory synaptic inputs, including EAA receptor-mediated inputs, but that under extreme conditions when all EAA receptors are blocked, an intrinsic mechanism generating autoactivity in these neurons is activated. It has also been shown that, in spontaneously hypertensive rats (11), Dahl salt-sensitive hypertensive rats (12), water-deprived rats (3), and in rats with renovascular hypertension (1), blockade of EAA receptors in the RVLM results in a significant decrease in MAP. It is therefore possible that EAA receptor-mediated inputs may contribute to the tonic activity of RVLM presympathetic neurons regulating RSNA under these abnormal conditions, but further studies are required to test this hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) of muscimol in the CVLM. These injections sites were estimated to be close to the level 13.4 mm caudal to bregma, but are drawn on to the nearest standard section from the atlas of Paxinos and Watson (
, centers of the sites of microinjections of Bic and vehicle in the control series of experiments.